Norepinephrine Drives Sleep Fragmentation Activation of Asparagine Endopeptidase, Locus Ceruleus Degeneration, and Hippocampal Amyloid-β42 Accumulation

Chronic sleep disruption (CSD), from insufficient or fragmented sleep and is an important risk factor for Alzheimer's disease (AD). Underlying mechanisms are not understood. CSD in mice results in degeneration of locus ceruleus neurons (LCn) and CA1 hippocampal neurons and increases hippocampal amyloid-β42 (Aβ42), entorhinal cortex (EC) tau phosphorylation (p-tau), and glial reactivity. LCn injury is increasingly implicated in AD pathogenesis. CSD increases NE turnover in LCn, and LCn norepinephrine (NE) metabolism activates asparagine endopeptidase (AEP), an enzyme known to cleave amyloid precursor protein (APP) and tau into neurotoxic fragments. We hypothesized that CSD would activate LCn AEP in an NE-dependent manner to induce LCn and hippocampal injury. Here, we studied LCn, hippocampal, and EC responses to CSD in mice deficient in NE [dopamine β-hydroxylase (Dbh)−/−] and control male and female mice, using a model of chronic fragmentation of sleep (CFS). Sleep was equally fragmented in Dbh−/− and control male and female mice, yet only Dbh−/− mice conferred resistance to CFS loss of LCn, LCn p-tau, and LCn AEP upregulation and activation as evidenced by an increase in AEP-cleaved APP and tau fragments. Absence of NE also prevented a CFS increase in hippocampal AEP-APP and Aβ42 but did not prevent CFS-increased AEP-tau and p-tau in the EC. Collectively, this work demonstrates AEP activation by CFS, establishes key roles for NE in both CFS degeneration of LCn neurons and CFS promotion of forebrain Aβ accumulation, and, thereby, identifies a key molecular link between CSD and specific AD neural injuries.


Introduction
Chronic sleep disruption (CSD) commonly occurs through chronic short sleep or chronic fragmentation of sleep (CFS).Both forms of CSD increase the risk of developing Alzheimer's disease (AD) and related dementias (Lim et al., 2013;Sabia et al., 2021).CSD increases neuronal activity-dependent amyloid-β (Aβ) peptide and tau production, while reducing glymphatic clearance of Aβ (J.E. Kang et al., 2009;Xie et al., 2013;Holth et al., 2019).Sleep disruption in young adult wildtype mice for 1 week imparts metabolic injury to the locus ceruleus neurons (LCn; J. Zhang et al., 2014), while extended sleep disruption in young adult mice manifests as lasting neural injury, including injury to and loss of LCn and CA1 hippocampal neurons, and increased hippocampal Aβ 42 , p-tau, and glial reactivity (Owen et al., 2021), supporting the concept that CSD can influence AD-vulnerable neuronal groups and influence Aβ and tau homeostasis, irreversibly and in the absence of a genetic predisposition to AD pathology.Yet, molecular mechanisms underlying the link between sleep loss and AD are not known.
LCn are of considerable interest in AD.Across the lifespan, LCn are the earliest neurons to accumulate hyperphosphorylated tau (Braak et al., 2011), and across Braak stages of AD, independent of age, LCn are progressively lost (Theofilas et al., 2017;Oh et al., 2019).Moreover, reduced integrity of the LC in humans and poor LC responsiveness to novelty (as measured with functional imaging) predict cognitive decline and/or greater amyloid plaque and tau tangle burden (Jacobs et al., 2021;Prokopiou et al., 2022).What we cannot ascertain from observational human studies is whether injury to the LCn contributes to any of the cognitive and neuropathology findings in AD.This question has begun to be addressed in a series of AD animal model studies.Lesioning LCn axons with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) accentuates some, but not all, features of tauopathy in the P301S mouse model (Chalermpalanupap et al., 2018).Similarly, DSP-4 increases gliosis and amyloid plaque deposition in mouse models of AD amyloid pathology (Heneka et al., 2006;Kalinin et al., 2007;Jardanhazi-Kurutz et al., 2010).These studies suggest that LCn injury may exacerbate Aβ and tau homeostasis, promoting neural injury.LCn are the primary source of norepinephrine (NE) to most areas of the brain, and NE is considered largely neuroprotective (O'Donnell et al., 2012).Thus, a question that remains unanswered following the LCn lesioning experiments is whether it is the loss of NE or axons or increased turnover of NE that contributes to the DSP-4 injury.
In support of the latter possibility, NE processing in LCn can result in the formation of toxic Aβ and tau fragments.Specifically, NE released from LCn may be taken back up into LCn varicosities or terminals and then metabolized by monoamine oxidase-A to 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), which may then activate asparagine endopeptidase (AEP) to cleave among other proteins amyloid precursor protein (APP) and tau (Zhang et al., 2015; S. S. Kang et al., 2020).The cleaved APP and tau fragments have been shown to promote Aβ 42 production and aggregation and tau mislocalization, aggregation, and propagation (Z.Zhang et al., 2014Zhang et al., , 2015; S. S. Kang et al., 2020Kang et al., , 2022)).
Considering the significance of LC injury and dysfunction in predicting AD outcomes, LCn vulnerability in both CSD and in AD and the large gaps in understanding the molecular mechanisms by which sleep disruption might influence AD, we sought to gain insight into the role of NE in the neural injuries previously observed in CSD that are also observed in AD.

Materials and Methods
Mice and study overview.Studies were performed at the University of Pennsylvania in accordance with the National Institutes of Health Office of Laboratory Animal Welfare Policy and the Institutional Animal Care and Use Committee.Dopamine β-hydroxylase (DBH) is required for NE biosynthesis, and NE is essential for fetal survival (Thomas et al., 1995).Female and male Dbh +/− mice with hybrid backgrounds of C57BL/6J and 129/SvCPJ.17were mated to generate Dbh −/− , Dbh +/− , and Dbh +/+ mice.Staged dams were then treated with 100 μg/ml phenylephrine and isoproterenol in their drinking water from embryonic days 8.5-16.5 and then with 2 mg/ml ʟ-threo-3,4-dihydroxyphenylserine (ʟ-DOPS, Lundbeck Pharmaceuticals), until their pups were born.Age-and sex-matched littermate Dbh +/− and Dbh +/+ mice were used as controls (Dbh + ) for Dbh −/− (Dbh − ), as tissue content of NE and epinephrine is normal in heterozygous mice, and phenotypic differences have not been observed relative to wild-type mice (Thomas and Palmiter, 1997).Mice were exposed to a 12 h light/dark exposure and were provided food and water ad libitum.At 10-12 months of age, mice were randomized to 16 weeks of CFS.A subset of mice underwent sleep recording studies to assess the effectiveness of sleep fragmentation prior and again 2 months into CFS.At 16-18 months of age, 2 months after CFS, mice were perfused for immunohistologic studies.
Sleep fragmentation protocol.CFS, which injures LCn (Li et al., 2014), was selected as our model of CSD.Mouse cages were placed atop an orbital rotor (MaxQ 2000) with the speed set at 120 rpm for 10 s/min, 24 h/d, controlled with an adjustable timer (H3CR-F8-300, Omron).Rested controls were housed in the same room without exposure to cage movement.Cages were modified with a vertical extender to allow water bottles with an elongated nozzle and ball valve to prevent water leakage.This method of sleep fragmentation does not elevate plasma corticosterone levels; mice maintain body weight, and the frequency of arousals in wild-type mice remains elevated across weeks of exposure (Li et al., 2014).
Electrode implantation and sleep recording and analysis.Dbh − and Dbh + mice were implanted with electroencephalographic (EEG) and electromyographic (EMG) electrodes for recording behavioral states.General anesthesia was induced with 3-4% isoflurane via mask and then maintained with 1-2.5% isoflurane.Using sterile procedures, two silver EEG electrodes (787000, A-M Systems) were placed bilaterally above the dorsal hippocampus, and reference electrodes were implanted over the rostral skull.Nuchal EMG electrodes were embedded in the dorsal nuchal musculature.Implanted electrodes were attached to a connector pedestal (MS363, Plastics One), which was secured with dental acrylic (C&B Metabond, Parkell).Mice recovered with littermates for 10 d and were then placed in individual cages for 3 d, prior to connecting the recording cable to a commutator (363 SL/6, SL6C Plastics One).Recordings were obtained under rested and CFS conditions at Week 8 CFS (n = 4-5/genotype).EEG and EMG signals were acquired at 256 Hz sampling frequency and amplified and filtered (EEG, 0.5-30 Hz and EMG, 1-100 Hz; 15A94 Grass Technologies).Data were recorded on AcqKnowledge3 software and analyzed as 4 s epochs in SleepSign (3.2, Kissei Comtec).Each epoch was scored as wake, nonrapid eye movement (NREM) sleep, or rapid eye movement (REM) sleep.Arousals were defined as the occurrence of one or more wake consecutive epochs following five or more consecutive sleep epochs (NREMS and/or REMS).The number of arousals/hour of sleep within the 24 h period is termed the arousal index, and this measure was used to characterize sleep fragmentation.Wake and NREMS bout numbers and durations were used as a secondary measure of sleep fragmentation.Because sleep has been described previously in Dbh − mice, additional sleep analyses were limited to pertinent outcomes: the arousal index, bout lengths and durations, and the percentage time in each stage/24 h, the latter to exclude confounds of chronic short sleep.
Histology, microscopy, and stereology.To examine the role NE plays in LCn, CA1, and EC responses to CFS, age-and sex-matched Dbh + and Dbh − mice exposed to rested or CFS conditions were transcardially perfused with 4% paraformaldehyde.Brains were postfixed overnight, cryopreserved, and then sectioned in a 1:6 series of 60 μm free-floating sections for specific immunohistological responses to sleep and genotype conditions.Details of the assessed primary antibodies for immunohistology are presented in Table 1.
We examined how NE influences the LCn survival response to CFS by carrying out stereology on Vector blue substrate (peroxidase-based, VectorLabs) labeled tyrosine hydroxylase (TH) positive neurons in pontine-caudal midbrain brain sections with Giemsa counter-staining.Five sections each spaced 120 μm and spanning bregma AP −5.20 to −5.65 mm (to represent the full rostral caudal extent of the LC nucleus) and matched across all mice were selected for n = 5-6 mice/sleep and genotype conditions.A Leica DM4B microscope equipped with a Stereo Investigator workstation (MicroBrightField) was used following an optical fractionator strategy.Our sampling scheme used a 0.25 x, y sampling frequency with a z-depth sampling of 0.80 to allow 2 μm guard zones for irregular surfaces and surface differences in immunolabeling on both sides.Previously, we validated this strategy and confirmed >150 counts/healthy adult mouse and Gundersen coefficients of error <0.10 (J.Zhang et al., 2014).Counters were blinded to genotype and sleep conditions.Giemsa-labeled nuclei within TH-labeled somata with clear Giemsa labeling of nuclear chromatin that came into focus within the x, y, and z-counting frame were counted in each section/mouse using a 100× magnification oil objective to calculate LCn counts/mouse in the software.
LC, CA1, and/or EC were then examined for genotype and sleep condition influences on asparagine endopeptidase (AEP), tau and APP products from AEP cleavage, phosphorylation of tau (p-tau), Aβ 42 , and microglia (Iba-1, CD68).One to three sections/mouse (n = 6-11 mice per genotype/sleep group) were processed with primary antibodies listed in Table 1 and appropriate secondary donkey anti-goat, anti-mouse, anti-rabbit, or anti-sheep antibodies labeled with Alexa Fluor 488, 594, or 647.Confocal microscopy (Leica SP5 AOBS) was used to image sections where laser intensity, exposure time, detector gain, amplifier offset, and depth of the focal plane within the section were standardized across compared image acquisitions.All image analyses were performed with scorers blinded to conditions using NIH ImageJ software by converting images to grayscale and measuring mean gray value over individual LCn (AEP, AEP-tau, AEP-APP) or by converting to 8 bit grayscale, inverting and measuring percentage area within a region of interest and using a standardized threshold for the image set.Immunofluorescent mean gray data were normalized to rested Dbh + values.
Statistical analysis.Statistical analyses were performed using GraphPad statistical software (Prism, versions 6.0 and 11.2).To examine how preselected behavioral state parameters varied with sleep conditions and genotype, a repeated measures analysis (within animal rested and CFS) was implemented.Analyses included assessment of an interaction effect between sleep condition and genotype (sleep × genotype) and/or main effects of sleep condition and genotype.Data were then compared across the sleep conditions and genotypes to determine the significance of group differences.To examine the effects of sleep condition and genotype on histopathologic variables, two-way ANOVA (full model with sleep condition, genotype, and sleep × genotype interaction) was used, with Tukey's multiple-comparisons post hoc (q) analyses.The cutoff for significant statistical power for all analyses was a multiplecomparisons corrected p value <0.05.

CFS-and NE-dependent AEP responses are evident in HC CA1
To ascertain how generalizable the CFS-induced increase in AEP is and its activation, we next examined AEP responses in the CA1 region of the hippocampus.In contrast with LCn genotype/sleep effects, there was no overall interaction, F (1,29) = 0.1, N.S., and no main genotype or sleep effect for CA1 AEP, F (1,29) = 3.5, N.S. and F (1,29) = 0.4, N.S., respectively (Fig. 4A,B).AEP, however, may be activated in the absence of protein upregulation by the presence of oxidative stress and/or an acidic environment (Zhao et al., 2014).To assess CFS effects on AEP activity, we next examined the effects of sleep disruption and Dbh genotype on AEP-tau in CA1.Both genotype and sleep main effects were observed (F (1,32) = 5.6; p < 0.05 and F (1,32) = 11.3;p < 0.01) without a sleep × genotype interaction (F (1,32) = 1.2, N.S.).Post hoc analyses revealed an increase in Dbh + mice from rested to CFS, q = 4.7; p < 0.05.AEP-tau was higher in CFS Dbh − mice than in rested Dbh + mice, q = 5.8; p < 0.05; p < 0.01 (Fig. 4A,C).For CA1 AEP-APP, a genotype/sleep interaction was found (F (1,30) = 6.5; p < 0.05) and a main sleep effect was observed (F (1,30) = 17.5; p < 0.001) without an overall genotype effect (F (1,30) = 2.9, N.S.).Specifically, AEP-APP was higher in CFS Dbh + mice than that in any of the other groups: rested Dbh + (q = 7.0; p < 0.001), rested Dbh − (q = 6.1; p < 0.001) and CFS Dbh − mice (q = 4.1; p < 0.05; Fig. 4A,D).In summary, without a measurable increase in AEP, CFS activates AEP in the HC, increasing both AEP-tau and AEP-APP in wild-type mice.The absence of an increase in AEP supports metabolic AEP activation in LCn by CFS.That Dbh − mice show increased AEP-tau in rested conditions suggests that AEP targeting of APP occurs independently from AEP targeting of tau in the HC, suggesting differences in subcellular localization.
Aβ 42 and tau phosphorylation responses are influenced by CFS and/or NE in CA1 and EC We have shown that chronic short sleep increases Aβ 42 in CA1 and increases p-tau (Ser202/Thr205, AT8) in both CA1 and the EC in wild-type mice (Owen et al., 2021).Here, we examined whether CFS influences p-tau and Aβ 42 in wild-type mice and whether NE influences the Aβ and p-tau responses to sleep disruption.A strong AT8 signal was evident in the EC, with negligible signal in the CA1 region, and thus measurements were obtained only in the EC.AT8 was measured in the EC across layers II-VI, and while a main genotype effect was not observed, F (1,19) = 0.5, N.S., a main sleep effect, F (1,19) = 4.5; p < 0.05, and a sleep × genotype interaction, F (1,19) = 10.0;p < 0.01, were observed.CFS increased EC AT8 in Dbh + mice relative to rested Dbh + mice, q = 5.4; p < 0.01 (Fig. 6A,C).There were no significant differences in AT8 across the other groups (q = 1.7-3.0,N.S.).was increased in CFS Dbh + mice, relative to the other three groups: rested Dbh + , q = 3.0; p < 0.05; rested Dbh − , q = 5.2; p < 0.01: and CFS Dbh − , q = 4.7; p < 0.05 (Fig. 6B,D).Aβ 42 colocalized with microglial cells labeled with Iba-1; however, there were no differences in Iba-1 percent area in CA1 across the four groups for an overall interaction, F (1,35) = 0.0, N.S.; main genotype, F (1,35) = 0.2, N.S.; or main sleep effect, F (1,35) = 0.5, N.S.An upregulation of CD68 suggests lysosomal activation in microglia and may be a more sensitive measure of microglial activation than Iba-1; thus, we next examined sleep and genotype effects on CA1 CD68.CD68 largely associated with microglia (Fig. 6B), and there were main sleep and genotype effects (F (1,28) = 7; p < 0.05 and F (1,28) = 19; p < 0.000) and a sleep × genotype interaction (F (1,28) = 8; p < 0.05).In Dbh + mice, CD68 increased in response to CFS (q = 5.3; p < 0.01), while levels were elevated in rested Dbh − mice relative to rested Dbh + mice (q = 7.1; p < 0.001) and did not increase further in response to CFS (q = 0.2, N.S.), as summarized in Figure 6F.In summary, as with the AEP-APP response in CA1, the CFS increase in CA1 Aβ 42 is dependent on NE.In contrast, transgenic absence of NE predisposes the mice to increased p-tau in CA1 and EC, in parallel with increased AEP-tau and microglial lysosomal activation.Collectively, these findings support the concept that

Discussion
Two general mechanisms have been proposed by which sleep disruption can perturb brain amyloid and tau homeostasis: increased production and/or release of Aβ and tau from wake-induced neuronal activation and reduced glymphatic clearance (Roh et al., 2012;Xie et al., 2013;Holth et al., 2019) LCn activation is important for arousal, and Dbh − mice show wake impairments at baseline (less wake time/24 h; Ouyang et al., 2004;Carter et al., 2010).Thus, one possibility for reduced injury in Dbh − mice could be reduced sleep fragmentation in Dbh − mice in response to CFS.However, in response to CFS, the two strains showed similar arousal indices, as well as similar numbers and lengths of NREM sleep bouts, supporting comparable fragmentation in the two strains.Only Dbh + mice evidenced reduced wakefulness/24 h in response to CFS.An impaired homeostatic response to sleep loss has been shown after LCn chemical lesioning (Cirelli et al., 2005), which may explain why CFS did not increase sleep drive in Dbh − mice.
The observation that sleep disruption activates AEP in LCn has important implications.AEP activation promotes p-tau, including phosphorylation at Ser202/Thr205, and in the P301S mouse model of tauopathy, absence of AEP prevents mutant taumediated CA1 synapse loss and cognitive dysfunction (Basurto-Islas et al., 2013;Z. Zhang et al., 2014).APP is also a substrate of AEP, and in mouse models of AD, deletion or inhibition of AEP normalizes synaptic function, improves cognitive function, and lessens Aβ and tau content and amyloid plaque accumulation (Zhang et al., 2015;Qian et al., 2024).Thus, AEP activation plays essential roles in neuronal injury and dysfunction in diverse AD mouse models, and here we show that CFS can activate AEP in key AD-vulnerable brain regions in the absence of AD genetic mutations and that this activation is dependent on NE.We suggest that the role of AEP in LCn and the forebrain in sleep loss effects on AD should now be examined for it roles in CSD and AD pathology and cognitive impairment.
Discordant AEP activation responses were observed in the forebrain for CFS-induced AEP cleavage of tau and APP.The distinct responses may in part be attributed to differences in subcellular localizations of these two AEP target proteins.APP is rapidly glycosylated and transported anterogradely within the trans-Golgi compartment directly to synapses, where APP positions as a transmembrane protein at synapses (Ferreira et al., 1993).APP is also identified in lysosomes and endosomes, presumably following endocytosis of synaptic membrane fragments with APP.Pro-AEP (the inactive precursor) translocates from the endoplasmic reticulum to lysosomes under conditions of cellular stress.The low pH in lysosomes converts pro-AEP to AEP, which then cleaves APP (Basurto-Islas et al., 2013).Activated AEP may also cleave synaptic transmembrane APP in the ectodomain to facilitate β-secretase cleavage to form Aβ 42 (Zhang et al., 2015).As the observed CFS-induced AEP-APP response was NE dependent, AEP-cleaved APP may originate in LCn synapses and varicosities within the hippocampus.In support, imaging revealed a punctate pattern for AEP-APP around CA1 neurons in the hippocampus, consistent with a synaptic source, but confirmation of subcellular localization will require electron microscopy or super-resolution microscopy.In contrast with APP's synaptic localization, tau normally localizes within axons as a microtubule-associated protein but can localize to the nucleus with age, where tau may function as a structural nucleic acid binding protein (Anton-Fernandez et al., 2023).Upon metabolic stress and phosphorylation, tau can migrate to the cell body and into dendrites (Ittner et al., 2010).In response to CFS in Dbh + mice, we observed AEP-tau within nuclei, somata, and dendrites of CA1 and EC neurons, supporting the concept that CFS results in metabolic stress in CA1 and EC pyramidal neurons, which in turn results in tau mislocalization and increased vulnerability to cleavage by AEP.The increase in p-tau in both the HC and EC in Dbh − mice was initially surprising but immediate early gene FosB is elevated in Dbh − mice, which upregulates cyclin-dependent kinase-5 (Chen et al., 2000), a key source of neuronal p-tau.
Molecular mechanisms of CSD injury to LCn are not well understood, yet the present work unveils a key driver.CSD imparts metabolic stress in LCn (J.Zhang et al., 2014), and in most experimental paradigms (including short sleep and CFS), CSD results in LCn loss (Shaffery et al., 2012;J. Zhang et al., 2014;Zhu et al., 2015;Deurveilher et al., 2021;Owen et al., 2021).LCn show increased firing frequencies across wake, particularly during exposures to novelty, relative to sleep, and show large bursts in firing rates upon arousals when autoreceptor tone is low in sleep (Aston-Jones and Bloom, 1981).Thus, short sleep and CFS are expected to increase NE release, reuptake, and metabolism in LCn.Here, we demonstrate that NE is necessary for CFS LCn loss.There are at least two mechanisms by which NE may contribute to LCn demise.The first is through AEP activation after NE is metabolized by monoamine oxidase-A to DOPEGAL (S. S. Kang et al., 2020Kang et al., , 2022)).AEP processing of tau and APP to toxic fragments may then contribute to neuronal demise.In support of NE-dependent AEP activation, CFS increased both AEP-tau and AEP-APP in LCn in Dbh + but not Dbh − mice.A second means by which NE may induce toxicity in LCn is through a monoamine oxidase-A-independent increase in oxidized quinones and reactive semiquinones (additional metabolites of NE), which may then promote depletion of glutathione and perturb functions of macromolecules that are critical to cell survival (Napolitano et al., 2011).NE-metabolized, quinone derivatives can also directly activate AEP (Santa-Maria et al., 2004;Bolton and Dunlap, 2017).Because NE serves numerous protective and adaptive roles in the brain, including roles in optimization of vascular, glial, and synaptic responses to increased neuronal activation (O'Donnell et al., 2012), and NE is necessary for appropriate sustained attention, mood, memory and stress responses (Aston-Jones and Bloom, 1981; Thomas and Palmiter, 1997;Sara, 2009;Gompf et al., 2010) and to regulate microglial homeostatic responses to locally increased neuronal activity (Liu et al., 2019;Stowell et al., 2019), and because increased NE within LCn appears deleterious to LCn, NE reuptake, and/or AEP inhibition, rather than NE receptor antagonism should be better options to explore as potential means to prevent CSD and AD neural injury.
In conclusion, NE plays a critical role in the LCn loss observed in response to CFS; NE is essential for CFS AEP upregulation and activation in LCn and the formation of toxic APP and tau fragments and the phosphorylation of tau in LCn, and NE is required for CFS activation of AEP in the HC and EC, as measured with AEP-cleaved APP and increased Aβ 42 in the HC.By identifying roles for LCn and NE in CFS forebrain Aβ dyshomeostasis, the findings provide both molecular and neuronal subtype windows into how sleep loss may influence AD pathogenesis.

Figure 1 .
Figure 1.Rotor platform rotation comparably fragments sleep in Dbh + and Dbh − mice.A, A representative polygraphic raw data image of an elicited arousal from nonrapid eye movement (NR) sleep.Top red tracing depicts the electromyographic (EMG) signal, and the white signal at the bottom shows the electroencephalographic (EEG) signal.The bottom panel bins EEG fast Fourier transform (FFT) power bins with blue representing delta or 0-4 Hz, gray for theta 6-9 Hz, and green for alpha, 11-14 Hz.The large white line marks the 10 s when the rotor platform is moving.The timing calibration bar is 4 s.B, Arousal index (number of arousals from sleep/total sleep time in hours) across two genotypes (Dbh + and Dbh − ) for two sleep conditions, rested, navy and chronic fragmentation of sleep, CFS, green/white).Shown are mean ± SE for n = 4/group.C-E, Twenty-four hour time (minutes) spent in each of the three behavioral states: wake (C), nonrapid eye movement sleep (NREMS, D), and rapid eye movement sleep (REMS, E) for Dbh + and Dbh − mice (n = 4/group, males and females balanced) analyzed with repeated measures two-way ANOVA and Tukey's post hoc testing.F, G, Number of wake (F) and NREMS (G) bouts/24 h in the same mice as for arousal index and behavioral state times.Values are mean ± SE.H, I, Duration of wake (H) and NREMS bout durations, as mean ± SE.In all graphs, navy represents baseline (rested) conditions and green/white diagonals represent CFS conditions.*p < 0.5; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

Table 1 .
Source and concentration details of primary antibodies used in experiments CFS upregulates and activates AEP in LCn in an NE-dependent mannerLCn in Dbh + and Dbh − mice exposed to rest or CFS conditions for 16 weeks were examined for AEP responses to CFS.Two sections/mouse (n = 7-11 mice/group) were analyzed.Males and females were balanced and combined in analysis.There were main genotype, F (1,31) = 13.4;p < 0.0001, and sleep condition effects, F (1,31) = 4.5; p < 0.05, on LCn AEP and an overall interaction F (1,31) = 5.9; p < 0.05.Specifically, Dbh + CFS mice had . Yet molecular mechanisms underlying sleep-related changes in production, release, and clearance are poorly understood, and little is known of why LCn are vulnerable in both sleep disruption and in AD.Here, we demonstrate that sleep disruption in the form of CFS induces LCn AEP activation with resultant production of AEP-cleaved APP and tau fragments in LCn, fragments known to be neurotoxic and contributory to AD pathology.We also show that NE is required for this CFS-induced AEP activation in the LC and for CFS loss of LCn.AEP activation by CFS was also evident in two AD-vulnerable forebrain regions, CA1 HC and the EC.In these regions, NE played very different roles for each of the two studied AEP targets, tau and APP, where NE deficiency increased AEP-tau and p-tau in a sleep-independent manner, while NE deficiency prevented the sleep-dependent (CFS) increase in AEP-APP and Aβ, demonstrating that NE critically determines the balance of Aβ and tau in AD-vulnerable brain regions across rested and sleep disruption conditions.